Molecular Genotyping of <i>Giardia duodenalis</i> Isolates from Symptomatic Individuals Attending Two Major Public Hospitals in Madrid, Spain

<div><p>Background</p><p>The flagellate protozoan <i>Giardia duodenalis</i> is an enteric parasite causing human giardiasis, a major gastrointestinal disease of global distribution affecting both developing and industrialised countries. In Spain, sporadic cases of giardiasis have been regularly identified, particularly in pediatric and immigrant populations. However, there is limited information on the genetic variability of circulating <i>G</i>. <i>duodenalis</i> isolates in the country.</p><p>Methods</p><p>In this longitudinal molecular epidemiological study we report the diversity and frequency of the <i>G</i>. <i>duodenalis</i> assemblages and sub-assemblages identified in 199 stool samples collected from 184 individual with symptoms compatible with giardiasis presenting to two major public hospitals in Madrid for the period December 2013–January 2015. <i>G</i>. <i>duodenalis</i> cysts were initially detected by conventional microscopy and/or immunochomatography on stool samples. Confirmation of the infection was performed by direct immunofluorescence and real-time PCR methods. <i>G</i>. <i>duodenalis</i> assemblages and sub-assemblages were determined by multi-locus genotyping of the glutamate dehydrogenase (<i>GDH</i>) and β-giardin (<i>BG</i>) genes of the parasite. Sociodemographic and clinical features of patients infected with <i>G</i>. <i>duodenalis</i> were also analysed.</p><p>Principal findings</p><p>Of 188 confirmed positive samples from 178 giardiasis cases a total of 124 <i>G</i>. <i>duodenalis</i> isolates were successfully typed at the <i>GDH</i> and/or the <i>BG</i> loci, revealing the presence of sub-assemblages BIV (62.1%), AII (15.3%), BIII (4.0%), AI (0.8%), and AIII (0.8%). Additionally, 6.5% of the isolates were only characterised at the assemblage level, being all of them assigned to assemblage B. Discordant genotype results AII/AIII or BIII/BIV were also observed in 10.5% of DNA isolates. A large number of multi-locus genotypes were identified in <i>G</i>. <i>duodenalis</i> assemblage B, but not assemblage A, isolates at both the <i>GDH</i> and <i>BG</i> loci, confirming the high degree of genetic variability observed in other molecular surveys. BIV was the most prevalent genetic variant of <i>G</i>. <i>duodenalis</i> found in individuals with symptomatic giardiasis in the population under study.</p><p>Conclusions</p><p>Human giardiasis is an ongoing public health problem in Spain affecting primarily young children under four years of age but also individuals of all age groups. Our typing and sub-typing results demonstrate that assemblage B is the most prevalent <i>G</i>. <i>duodenalis</i> assemblage circulating in patients with clinical giardiasis in Central Spain. Our analyses also revealed a large genetic variability in assemblage B (but not assemblage A) isolates of the parasite, corroborating the information obtained in similar studies in other geographical regions. We believe that molecular data presented here provide epidemiological evidence at the population level in support of the existence of genetic exchange within assemblages of <i>G</i>. <i>duodenalis</i>.</p>